Potassium channels are tetrameric membrane proteins that provide a highly selective conduit for potassium ions to diffuse across the hydrophobic barrier of cell membranes. As such, their formation and biophysical properties are critical for processes like neuronal excitability, secretion of hormones, and muscle contraction. The formation of ion channels includes biogenesis of monomeric channel subunits, assembly of subunits into the quaternary channel, and trafficking of the channel to the appropriate cellular membrane where it performs its functional role. Although the structure and function of mature potassium channels have been studied extensively, little is known about the early folding events in channel biogenesis. Defects in translation and folding have consequences for assembly, trafficking, and function of potassium channels, and underlie pathology. This proposal focuses on folding events of various domains of the voltage-gated potassium (Kv) channel. We have developed several biochemical approaches to define the stages and compartments in which secondary, tertiary, and quaternary structures of Kv channels are acquired. The initial steps in biogenesis of Kv proteins require translation of the mRNA on the ribosome. Thus, we study folding events while the nascent Kv peptide is attached to the ribosome as a biogenic intermediate. The results of our studies bear not only on Kv channel formation and cellular levels of Kv protein, but also on broader issues in biogenesis and folding of proteins. This is the overall goal of our proposal. This goal is divided into four specific aims. The first is devoted to understanding subdomain formation of the cytosolic T1 domain of Kv channels. The second aim is devoted to elucidating folding events of components of the voltage-sensing domain. The third aim will define biogenesis of the pore region, and the fourth will determine the role of the ribosomal tunnel in Kv folding. Many of the methods and strategies used in these proposed studies (e.g., pegylation, intramolecular crosslinking assays of a nascent peptide attached to a ribosomal tunnel) were introduced and developed in our laboratory to assay secondary, tertiary, and quaternary folding events. The proposed experiments will use a range of techniques including these novel biochemical micro-assays of Kv biogenic intermediates and electrophysiological assays of Xenopus oocytes. PUBLIC HEALTH RELEVANCE: The biogenesis and biochemical processing of voltage-gated potassium channels are critical to their functional activity in a cell. These processes include synthesis of the channel protein, assembly of these proteins into multimeric channels, and delivery of these channels to the plasma membrane. Defects in any of these steps will result in altered expression of the channels at the cell surface, with pathological and often lethal consequences. Our studies of ion channel folding bear on basic principles of protein folding and pave the way for understanding the molecular basis of protein misfolding, a fundamental cause of many diseases.